High levels of LDL are a major risk factor for coronary disease and are the source for most of the cholesterol that accumulates in the arterial wall (Ross, R. 1995. Annu. Rev. Physiol. 57:791-804). Subendothelial retention of LDL has been suggested to be a key pathogenic process in atherosclerosis, and several lines of circumstantial evidence suggest that intramural retention of atherogenic lipoproteins involves the extracellular matrix, chiefly proteoglycans (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc. Biol. 15:551-561; and Radhakrishnamurthy, B. et al. 1990. Eur. Heart J. 11 Suppl E: 148-157).
The significance of the possible LDL proteoglycan interaction has been highlighted in two recent review articles (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; and Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc Biol. 15:551-561). Williams and Tabas proposed that subendothelial retention of atherogenic lipoproteins is the central pathogenic process in atherosclerosis. Moreover, they hypothesized that retained lipoproteins can directly or indirectly provoke all known features of early lesions, such as lipoprotein oxidation, monocyte migration into the artery wall, macrophage foam cell formation, and cytokine production, and can accelerate further retention by stimulating local synthesis of proteoglycans. Several lines of evidence indicate that the retention of arterial lipoproteins involves the extracellular matrix; proteoglycans in particular have been hypothesized to play an important role (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc Biol. 15:551-561; Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; and Hurt, E., and G. Camejo. 1987. Atherosclerosis. 67:115-126). First, purified arterial proteoglycans, especially those from lesion-prone sites (Cardoso, L. E., and P. A. Mourao. 1994. Arterioscler. Thromb. 14:115-124; and Ismail, N. et al. 1994. Atherosclerosis. 105:79-87), bind atherogenic lipoproteins in vitro, particularly LDL from patients with coronary artery disease (Linden, T. et al. 1989. Eur. J. Clin. Invest. 19:38-44). LDL binds with high affinity to dermatan sulfate and chondroitin sulfate proteoglycans produced by proliferating smooth muscle cells (Camejo, G. et al. 1993. J. Biol Chem. 268:14131-1437). Second, proteoglycans are a major component of the artery wall extracellular matrix and are available to participate in the interactions of lipoproteins in the earliest stages of atherogenesis. Third, retained apo-B immunologically co-localizes with proteoglycans in early and developed lesions (Walton, K., and N. Williamson. 1968. J. Atheroscler. Res. 8:599-624; Hoff, H., and G. Bond. 1983. Artery. 12:104-116; Hoff, H. F., and W. D. Wagner. 1986. Atherosclerosis. 61:231-236; Nievelstein-Post, P. et al. 1994. Arterioscler. Thromb. 14:1151-1161; and Galis, Z. et al. 1993. Am J. Pathol. 142:1432-1438). The observation that the arterial wall content of these proteoglycans increases during atherosclerosis and correlates with an increased accumulation of aortic cholesterol also supports the potential importance of the interaction between LDL and proteoglycans (Hoff, H. F., and W. D. Wagner. 1986. Atherosclerosis. 61:231-236; Merrilees, M. et al. 1990. Arteriosclerosis. 81:245-254).
Proteoglycans contain long carbohydrate side-chains of glycosaminoglycans, which are covalently attached to a core protein by a glycosidic linkage. The glycosaminoglycans consist of repeating disaccharide units, all bearing negatively charged groups, usually sulfate or carbohydrate groups. In vitro, LDL bind with high affinity to many proteoglycans found in the artery wall, including dermatan sulfate proteoglycans (e.g., biglycan) and chondroitin sulfate proteoglycans (e.g., versican), which are produced by smooth muscle cells in response to PDGF or TGFβ (Schonherr, E. et al. 1991. J. Biol. Chem. 266:17640-17647; and Schönherr, E. et al. 1993. Arterioscler. Thromb. 13:1026-1036). The interaction between LDL and proteoglycans have been hypothesized to involve clusters of basic amino acids in apo-B100, the protein moiety of LDL, that interact with the negatively charged glycosaminoglycan proteoglycans (Mahley, R. et al. 1979. Biochem. Biophys. Acta. 575:81-91; Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512) or by bridging molecules such as apo-E or lipoprotein lipase (Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc. Biol. 15:551-561).
Isolation of large fragments of apo-B100 from different regions characterized by concentrations of positive clusters indicated that up to eight specific regions in apo-B100 bind proteoglycans (Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512). Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103 identified two fragments, residues 3134-3209 and 3356-3489, that bind to heparin with the highest affinity. Recently Camejo and coworkers confirmed this finding and proposed that residues 3147-3157 and 3359-3367 may act cooperatively in the association with proteoglycans (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; and Olsson, U. et al. 1997. Arterioscler. Throm. Vasc. Biol. 17:149-155). However, because these studies were carried out with delipidated apo-B fragments in the presence of urea or with short synthetic apo-B peptides, it is not clear which of the binding sites are functionally expressed on the surface of LDL particles. Some or many of these postulated glycosaminoglycan-binding sites may not be functional when apo-B is associated with LDL. For example, apo-E has two heparin-binding sites, but only one binds to heparin when apo-E is completed with phospholipid (Weisgraber, K. et al. 1986. J. Biol Chen 261:2068-2076). This heparin-binding site coincides with the LDL receptor-binding site of apo-E.
Although eight potential glycosaminoglycan-binding sites have been identified in apo-B100 (Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512), it was not known which of them participate in the physiological binding of LDL to proteoglycans. Previously, we have demonstrated, in conjunction with others, that Site B (residues 3359-3369) is the LDL receptor-binding site, and in the study which generated the present invention we found that it is also the primary binding site to proteoglycans.
Modification of LDL potentially exposes the other proteoglycan-binding sites. Paananen and Kovanen (Paananen, K., and P. T. Kovanene. 1994. J. Biol. Chem. 269:2023-2031) noted that proteolysis of apo-B100 strengthened the binding of LDL to proteoglycans, suggesting the exposure of buried heparin binding sites. Likewise, when LDL are fused by sphingomyelinase treatment, the modified lipoproteins bind more avidly to proteoglycans. The finding that multiple heparin molecules bind to LDL (Cardin, A. et al. 1987. Biochemistry. 26:5513-5518) may also be explained by a cooperative effect of heparin binding to one site that triggers a conformational change in apo-B100 that enables other sites to participate in the interaction. Thus, the initial interaction with proteoglycans may induce structural alterations of the LDL that expose heparin/proteoglycan-binding sites that may contribute to the intramural retention of LDL after the initial interaction with the primary binding site.
The interaction between LDL and the LDL receptor plays a major role in determining plasma cholesterol levels in humans and other mammalian species (Goldstein, J. et al. 1985. Annu. Rev. Cell Biol. 1:1-39). Apo-B100 is the major protein component of LDL and is responsible for the binding of these lipoproteins to the LDL receptor (Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349). The relevance of this catabolic pathway is best illustrated by the genetic disorders familial hypercholesterolemia (FH) and familial defective apo-B100 (FDB), in which high levels of LDL accumulate in the circulation because mutations in the LDL receptor (FH) or in the ligand (FDB) disrupt the binding of LDL to its receptor (Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349). Many different mutations of the LDL receptor cause FH (Hobbs, H. et al. 1992. Hum. Mutat. 1:445-466), but FDB is associated with a single site mutation, the substitution of glutamine (Innerarity, T. et al. 1987. Proc. Natl. Acad. Sci. USA. 84:6919-6923) or, in a few cases, tryptophan (Gaffney, D. et al. 1995. Arterioscler. Thromb. Vasc. Biol. 15:1025-1029) for the normally occurring arginine at residue 3500 of apo-B100. With the exception of an arginine-3531 to cysteine mutation (Pullinger, C. et al. 1995. J. Clin. Invest. 95:1225-1234), which is associated with a minor decrease in LDL receptor binding, extensive searches have not found any other mutations of apo-B100 that cause defective receptor binding of LDL (Pullinger, C. et al. 1995. J. Clin. Invest. 95:1225-1234). The FDB mutation occurs at an estimated frequency of 1/500 in the normal population and is therefore one of the most common known single-gene defects causing an inherited abnormality (Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349).
Much attention has focused on understanding the molecular interaction between apo-B100 and the LDL receptor. The structural and functional domains of the LDL receptor have been defined in detail (Hobbs, H. et al. 1992. Hum. Mutat. 1:445-466), but much less is understood about the receptor-binding domain of apo-B100, because of its large size and insolubility in aqueous buffer. Furthermore, both the lipid composition and the conformation of apo-B100 appear to be crucial to its function as an effective ligand for the LDL receptor, since apo-B100 binds to the LDL receptor only after the conversion of large VLDL to smaller LDL (Goldstein, J. et al. 1985. Annu. Rev. Cell Biol. 1:1-39).
Selective chemical modification of the apo-B100 of LDL demonstrated that the basic amino acids arginine and lysine were important in the interaction of LDL with its receptor (Mahley, R. et al. 1977. J. Biol. Chem. 252:7279-7287; and Weisgraber, K. et al. 1978. J. Biol. Chem. 253:9053-9062). Once apo-B100 was sequenced, several regions enriched in arginine and lysine residues became candidates for receptor binding, including Site A (residues 3147-3157) and Site B (residues 3359-3367) (Knott, T. et al. 1985. Science. 230:3743).
While it had been hypothesized that LDL-proteoglycan binding was possibly important to the formation of atherosclerotic lesions through the retention of lipoproteins in the subendothelium, this hypothesis has not been empirically demonstrated in the art. Moreover, there have been six obstacles which have prevented other researchers from demonstrating the mechanism by which atherogenesis occurs and using this information to combat atherosclerosis. First, there have been eight potential sites identified in the apo-B100 protein, any one or several of which could have been responsible for proteoglycans trapping LDL in the subendothelium. Second, it has been unknown which potential sites in the apo-B100 are exposed to the surface of the LDL particles and which are buried within the lipid core. Third, there has been evidence that some of the potential proteoglycan binding sites on apo-B100 may work cooperatively, creating the possibility that blocking proteoglycan binding at any single site might not have proven both necessary and sufficient to eliminate LDL retention in the subendothelium. Fourth, the modification of LDL has been shown in some cases to expose new proteoglycan binding sites to the surface. Fifth, any disruption to LDL proteoglycan binding had the potential to disrupt LDL receptor binding, which would serve to disrupt the natural clearance of LDL from blood, raise serum cholesterol levels, and potentially result in a condition similar to familial hypercholesterolemia. Sixth, it has not been possible to use site-directed mutagenesis and express the entire mutated apo-B100 proteins as LDL in order to define the proteoglycan-binding sites on LDL.